Fructophilic lactic acid producing bacteria

ABSTRACT

The present invention discloses a novel fructophilic lactic acid producing bacteria Bacillus coagulans strain FF-7 (MTCC 25235) and the process of isolation and characterization of the bacteria. The invention also discloses the biological applications/therapeutic use of fructophilic lactic acid producing bacteria in increased utilization of fructose from food stuff and in the managing disorders related to high fructose intake.

BACKGROUND OF THE INVENTION Field of the Invention

The invention in general relates to fructophilic lactic acid producingbacteria. More specifically, the present invention relates to isolation,characterization and biological applications of fructophilic probioticbacteria Bacillus coagulans.

Description of Prior Art

Probiotics and its importance in health and prevention of many disordershave been already reported. Fructophilic lactic acid bacteria (FLAB) area special group of lactic acid bacteria which utilizes fructose as agrowth substrate. Due to their unique characteristics of poor growth onglucose and preference of oxygen, they are regarded as “unconventional”lactic acid bacteria (LAB). Their unusual growth characteristics are dueto an incomplete gene encoding a bifunctional alcohol/acetaldehydedehydrogenase (adhE). This results in the imbalance of NAD/NADH and therequirement of additional electron acceptors to metabolize glucose.Oxygen, fructose, and pyruvate are used as electron acceptors. FLAB havesignificantly fewer genes for carbohydrate metabolism than other LAB,especially due to the lack of complete phosphotransferase system (PTS)transporters. FLABs were originally classified as Leuconostoc speciesand later reclassified as Fructobacillus species based on theirphylogenetic positions and biochemical and morphologicalcharacteristics. (Endo A, Okada S. 2008. Reclassification of the genusLeuconostoc and proposals of Fructobacillus fructosus gen. nov., comb.nov., Fructobacillus durionis comb. nov., Fructobacillus ficulneus comb.nov. and Fructobacillus pseudoficulneus comb. nov. Int J Syst EvolMicrobiol 58:2195-2205).

The metabolism of fructose starts with the enzyme fructase in the liver.The fructose load is converted into lactate in enterocytes and in theliver. Further, excess fructose in the liver is directed towardperipheral tissues, and is taken up by the insulin-dependent glucosetransporter, GLUT 4, present on adipose tissue is converted into fattyacids. GLUT 4 is reported to play an important role in the developmentof fructose-induced hepatic steatosis and dyslipidemia. Further, severalfructose induced metabolic disorders are reported like NASH, NAFLD,Dyslipidemia, ectopic lipid deposition in the liver and skeletal muscle,Uric Acid Metabolism, High Blood Pressure, Mineral Metabolism.(Prasanthi Jegatheesan and Jean-Pascal De Bandt, Fructose and NAFLD: TheMultifaceted Aspects of Fructose Metabolism, Nutrients. 2017 Mar; 9(3):230; Bidwell A J, Chronic Fructose Ingestion as a Major Health Concern:Is a Sedentary Lifestyle Making It Worse? A Review, Nutrients. 2017 Jun;9(6): 549). Excessive fructose intake is also associated with increasedcardiovascular risk. Increase in fructose intake may also lead to lacticacidosis by excessively decreasing the pH in blood.

Probiotics play an important role in metabolizing intestinal fructose.The low dose of fructose that is orally consumed, reach the intestineand is metabolized in the presence of different enzymes and inherentmicrobiota. However, in the presence of high dose of fructose, the loadof fructose spills over from intestine to liver. In order to reduce thespillover of fructose from intestine to liver, probiotics play animportant role to convert fructose to SCFAs (Short chain fatty acids).

FLABs are isolated from various sources such as fruits and vegetables,human stool culture, natural antimicrobial agent, cheese, kefir grains,dairy and non-dairy products, fermented and raw milk, feces of breastfed infants, lactating milk, sheep, buffalo and cow milk, yogurt,beverages, poultry sources, animal rumen contents, Pengging Duck'scaecum, chicken intestine and fecal samples, chicken feed, enzymes,fermented rice, curd, meat and yeast extracts, glucose and sucrose,human gut, human colonial epithelial cells, human and animal vagina andmouth extraction, diapers of human babies, pineapples wastes, industrialsausages, ice-cream, small intestines of piglets, corn slurry, crop andintestinal ducks. The following prior art documents describes theisolation of different FLABs.

1. Akihito Endo, Fructophilic lactic acid bacteria inhabit fructose-richniches in nature, Microb Ecol Health Dis. 2012; 23

2. Akihito Endo, Shintaro Maeno, Yasuhiro Tanizawa, Wolfgang Kneifel,Masanori Arita, Leon Dicks, Seppo Salminen, Fructophilic Lactic AcidBacteria, a Unique Group of Fructose Fermenting Microbes, Minireview,Applied and Environmental Microbiology, October 2018 Volume 84 Issue 19e01290-18

3. Arshad F, Mehmood R, Hussain S, Khan M A, Khan M S (2018)Lactobacilli as Probiotics and their Isolation from Different Sources.Br J Res 5 (3): 43

Given that the biological effects of probiotics are strain specific andcannot be generalized to all strains and species (Guidelines for theevaluation of probiotics in food, Joint FAO/WHO Working Group Report onDrafting Guidelines for the Evaluation of Probiotics in Food, London,Ontario, Canada, Apr. 30 and May 1, 2002, See section 3.1 indicatingthat “The current state of evidence suggests that probiotic effects arestrain specific. Strain identity is important to link a strain to aspecific health effect as well as to enable accurate surveillance andepidemiological studies.”), there still exists a need to find a superiorfructophilic probiotic bacteria with improved biological functions. Thepresent invention solves the above problem by disclosing a novelfructophilic lactic acid bacteria Bacillus coagulans MTCC 25235.

It is the principal objective of the invention to disclose a novelfructophilic lactic acid producing bacteria Bacillus coagulans MTCC25235 and its process of isolation.

It is another objective of the invention to disclose the production ofshort chain fatty acid by fructophilic lactic acid producing bacteriaBacillus coagulans MTCC 25235.

It is yet another objective of the invention to disclose theanti-microbial effects of fructophilic lactic acid producing bacteriaBacillus coagulans MTCC 25235.

The present invention solves the above mentioned objectives and providesfurther related advantages.

Deposit of Biological Material

The deposit of biological material Bacillus coagulans strain FF7 bearingaccession number MTCC 25235, mentioned in the instant application hasbeen made on 28 Feb. 2019 at Microbial Type Culture Collection & GeneBank (MTCC), CSIR-Institute of Microbial Technology, Sector 39-A,Chandigarh—160036. India.

SUMMARY OF THE INVENTION

The present invention discloses the fructophilic lactic acid producingbacteria. The invention further discloses the process of isolation,characterization and biological applications/therapeutic use offructophilic lactic acid producing bacteria.

Other features and advantages of the present invention will becomeapparent from the following more detailed description, which illustrate,by way of example, the principle of the invention.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the office upon request and paymentof the necessary fee.

FIGS. 1A, 1B, 1C and 1D shows the phase contrast microscopic image(×1000 magnification) of Bacillus coagulans MTCC 25235 (FIG. 1A), wetmount of cells with the spores (FIG. 1B), Gram staining of vegetativecells (FIG. 1C), Spore staining of sporulated cells and (FIG. 1D),colony grown on GYE agar plate.

FIG. 2 shows graphical representation of comparison of growth pattern ofBacillus coagulans FF7, MTCC 25235 and B. coagulans ATCC 31284 inpresence of fructose and dextrose as carbon source. Values are mean(±SD) from three independent determinations.

FIG. 3 shows graphical representation of utilization of fructose byBacillus coagulans MTCC FF7, 25235 after 24 h of incubation in presenceof media and fructose rich foods (Honey, fruit juice). Values are mean(±SD) from three independent determinations.

FIG. 4 shows phylogenetic tree based on the sequence of 16S rRNA showingthe relative positions of Bacillus coagulans FF7.

FIG. 5 shows effect of different pH on the growth of B. coagulans FF7,MTCC 25235 and B. coagulans ATCC 31284, optimum pH for the growth wasfound to be pH 7.5 and pH 6.5 respectively. Values are mean (±SD) fromthree independent determinations.

FIG. 6 shows graphical representation of effects of differenttemperatures on the growth of B. coagulans MTCC FF7, 25235 and B.coagulans ATCC 31284. Values are mean (±SD) from three independentdeterminations.

FIG. 7 shows Effect of GIT hostile conditions on the viability of B.coagulans MTCC 25235 after gastric treatment in an in-vitro experimentmimicking invivo conditions. Sterile saline was taken as untreatedcontrol.

FIGS. 8A and 8 b shows BSH activity of Bacillus coagulans MTCC 25235.(FIG. 8A) Determination using soft MRS agar supplemented with ox bile(0.3%, w/v) and CaCO3 (0.3%, w/v) and MRS agar without bile salts servedas the negative control (FIG. 8B). Hollow zones suggest the BSH activityof B. coagulans MTCC 25235.

FIG. 9 shows graphical representation of the survival of Bacilluscoagulans MTCC FF7, 25235 at various pH values (1.5-8.0) in gastricjuice buffer. The values are expressed in Log10 spores/g. Data representthe mean and standard deviations (±SD) of two different experimentsperformed.

FIG. 10 shows graphical representation of in-vitro effect of ox bilesalt on the growth of B. coagulans FF7, MTCC 25235 and B. coagulans ATCC31284. The overnight grown fresh culture of B. coagulans MTCC 25235 andB. coagulans ATCC 31284 were inoculated in MRS broth with (0.1, 0.3,0.4, 0.5, 0.6, 0.8, 0.9 and 1%, w/v) and without ox bile salt (%, w/v).Values are mean (±SD) from three independent determinations.

FIG. 11 shows graphical presentation of production of L-Lactic acid byB. coagulanus FF7, MTCC 25235 in presence of two standardizedpreparations equivalent to 6×10⁹ (preparation 1) and 15×10⁹ cfu/g(preparation 2) were studied. Values are mean (±SD) from threeindependent determinations.

FIGS. 12A, 12B and 12C shows graphical presentation of Acetic FIG. 12A,butyric FIG. 12B and propionic acid FIG. 12C production from theBacillus coagulans MTCC 25235 while fermenting fructose, FOS, cranberryseed fibre, fenugreek seeds fibre. Samples were collected after 4, 8,12, 18 and 24 h of fermentation. Values are average mean of triplicateperformed at two different occasions and represented in mg/g.

FIG. 13 shows graphical presentation of Viability of B. coagulans MTCC25235 during the storage at temperature 40±2° C. with RH 60% ±5%. Twostandardized preparations equivalent to 15×10⁹ (preparation 1) and 6×10⁹cfu/g (preparation 2) were studied. Average means of spore viable countsare expressed in log10 cfu/g. Each time point represents the mean Log₁₀standard deviations (±SD) of three different experiments performed induplicate.

DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS

In the most preferred embodiment, the present invention discloses amethod for isolating and identifying novel fructose utilizing probioticbacteria from honey, comprising step of:

-   -   a) Mixing honey with saline in the ratio of 1:10 w/v to obtain a        suspension;    -   b) Thoroughly mixing the suspension of step a) and providing        heat shock at 50-70° C. for 30 minutes for selective isolation        of spores;    -   c) Isolating the bacterial colonies by incubating 1-2 ml of the        suspension from step b) in suitable culture media containing        fructose for 48 hrs at 35-37° C.;    -   d) Purifying bacterial isolates by selecting and incubating        morphologically distinct colonies in a suitable media containing        fructose as carbon source;    -   e) Identifying the bacterial strain by biochemical analysis and        16S rRNA sequencing as Bacillus coagulans strain FF7 bearing        accession no. MTCC 25235.

In a related aspect, the honey is selected from the group comprising,but not limited to, raw honey, filtered honey, acacia honey, alfalfahoney, aster honey, avocado honey, basswood honey, beechwood honey,blueberry honey, bluegum honey, buckwheat honey, clover honey, dandelionhoney, eucalyptus honey, fireweed honey, heather honey, ironbark honey,jarrah honey, leatherwood honey, linden honey, macadamia honey, manukahoney, orangeblossom honey, pinetree honey, sourwood honey, sage honey,and tupelo honey. In another related aspect, the culture media isselected from the group comprising MRS (De Man, Rogosa and Sharpe agar),GYA (Glucose Yeast Extract Agar), TSB (Tryptone Soya Broth), Sporulationmedia and Mueller Hinton Agar.

In another related aspect, the isolated probiotic strain returnspositive for biochemical tests catalase, oxidase, methyl red, vogesproskauers, lactose, xylose, maltose, fructose, dextrose, galactose,raffinose, trehalose, melobiose, sucrose, arabinose, mannose, inulin,sodium gluconate, salicin, sorbitol, mannitol, arabitol, methylglucoside, rhamnose, cellobiose, ONPG, esculin hydrolysis and negativefor biochemical tests sorbose malonate utilization, citrate utilization,xylitol, methyl mannoside, melezitose, erythritol, adonitol, inositol,dulcitol, glycerol, blood hemolysis, citrate and indole.

In another preferred embodiment, the invention discloses a novelprobiotic bacteria of the genus Bacillus, isolated from honey forincreased utilization of fructose from foods rich in fructose. Inanother related aspect, the fructophilic probiotic bacterium is Grampositive. In yet another aspect, optimum pH and temperature recorded forthe growth of fructophilic bacteria is 7.5 and 40° C. respectively. Inanother aspect, the fructophilic probiotic bacteria is bile tolerant,gastric acid resistance and produces lactic acid. In a related aspect,the fructophilic probiotic bacteria is Bacillus coagulans. In yetanother related embodiment, the Bacillus coagulans strain is Bacilluscoagulans MTCC 25235. In a related aspect, the foods rich in fructoseare selected from the group comprising high fructose corn syrup, honey,Agave, Maple syrup, Coconut sugar, Palm sugar, Molasses, Soda, Candies,sweetened yogurt, frozen foods, canned foods, cereals, fruit juices,coffee creamer, jams and jellies, energy drinks, condiments, ice cream.In a related aspect, the fructophilic probiotic bacteria is used for thetherapeutic management of disorders related to high fructose intake. Ina related aspect, diseases related to high fructose intake are selectedfrom the group comprising, but not limited, obesity, non-alcoholicsteatohepatitis (NASH), insulin resistance, metabolic syndrome,cardio-vascular complications, diabetes, hyperlipidemia, hypertension,inflammation and hyperuricemia. In another related aspect, thefructophilic probiotic bacteria is present in the form of an inoculum,freeze-dried powder, fine powder, tablet, capsule, suspension, solution,emulsion, gummy, chewable or edible foods and administered as astand-alone or in combination with foods rich in fructose selected fromthe group comprising high fructose corn syrup, honey, Agave, Maplesyrup, Coconut sugar, Palm sugar, Molasses, Soda, Candies, sweetenedyogurt, frozen foods, canned foods, cereals, fruit juices, coffeecreamer, jams and jellies, energy drinks, condiments, ice cream.

In yet another preferred embodiment, the invention discloses a method ofinhibiting pathogenic microbes said method comprising step of bringingto contact said microbes with the fructophilic probiotic bacteriaBacillus coagulans MTCC 25235. In a related aspect, the pathogenicmicrobes are selected from the group comprising Salmonella abony,Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa, Bacilluscereus, Propionibacterium acnes, Streptococcus mutans, Staphylococcusaureus, Staphylococcus epidermidis.

In another preferred embodiment, the invention discloses a method ofproducing short chain fatty acids by culturing the fructophilicprobiotic bacteria Bacillus coagulans MTCC 25235 along with plant fibersselected from the group consisting of fructose, fenugreek seed fibers,cranberry seed fibers, fructooligosaccharides (FOS).

The specific examples included herein below illustrate the aforesaidmost preferred embodiments of the present invention.

EXAMPLES Example 1 Isolation and Identification of Fructophilic BacteriaMethods

Raw unfiltered honey was used in this study to isolate the spore formingfructophilic lactic acid bacteria. For the isolation of fructophilicbacteria, de Man, Rogosa and Sharpe (MRS) (dextrose replaced byfructose) was used in this study (table 1).

TABLE 1 Media composition Sr. No. Ingredients Gms/Liter 1 Proteosepeptone 10.000 2 Beef extract 10.000 3 Yeast extract 5.000 4 Fructose20.000 5 Polysorbate 80 1.000 6 Ammonium citrate 2.000 7 Sodium acetate5.000 8 Magnesium sulphate 0.100 9 Manganese sulphate 0.050 10Dipotassium phosphate 2.000 11 Agar 12.000 12 Final pH (at 25° C.) 6.5 ±0.2

One gm of unfiltered raw honey was taken into the test tubes containing10 ml of saline. This was mixed well and heat shock was given at 70° C.for 30 minutes for selective isolation of spores. Isolation of bacteriawas carried out by adding 1 ml of above sample from each dilution ontothe MRS agar plates containing fructose. Plates were further incubatedat 37° C. for 48 h. After incubation, morphologically distinct colonieswere picked up for further testing and purification of bacterialisolates. Bacillus coagulans MTCC 25235 was isolated and streaked onother MRS agar plate containing fructose as carbon source.

Biochemical Characterization of Fructophilic Bacteria

Bacterial isolates were grown in de Man, Rogosa and Sharpe Agar (MRSA)for 24 h at 37° C. Bacterial inoculums were prepared by picking 1 to 3well isolated colonies and a homogenous suspension in sterile saline wasprepared. The density of suspension was ≥0.5 OD at 620 nm. This inoculum(50 μl) of was used and test was performed as per the kit manufacturer'sinstructions (HiMedia, Mumbai, India). Biochemical characterization ofBacillus coagulans MTCC 25235 and B. coagulans ATCC 31284 was performedby the method described (Majeed, M., Nagabhushanam, K., Natarajan, S.,Sivakumar, A., Eshuis-de Ruiter, T., Booij-Veurink, J., JanineBooij-Veurink, Ynte P. de Vries, Ali, F. (2016); Evaluation of geneticand phenotypic consistency of Bacillus coagulans MTCC 5856: A commercialprobiotic strain. World Journal of Microbiology & Biotechnology, 32,60).HiCarbohydrate™ Kit (code-KB009) was procured from HiMedia, Mumbai,India and tests were performed as per the manufacturer's instructions.Bacterial suspension of Bacillus coagulans MTCC 25235 and B. coagulansATCC 31284 was prepared as mentioned in above section. Further, IMViC(indole, methyl red, Voges Proskauer, Citrate utilization) test, oxidaseand Gram staining of and B. coagulans ATCC 31280 were performed as perthe method Majeed et al., (2016); (Cappucino G and Natalie Sherman.Microbiology: A Laboratory manual 5th edition. 1998).

16S rDNA Sequencing

16S rDNA sequencing Genomic DNA of the B. coagulans MTCC 5856 wasprepared as previously described by (William J. Bruno, Nicholas D.Socci, and Aaron L. Halpern (2000). Weighted Neighbor Joining: ALikelihood-Based Approach to Distance-Based Phylogeny Reconstruction,Mol. Biol. Evol. 17 (1): 189-197). A fragment of the 16S rDNA gene wassequenced using an ABI 3500 genetic analyser automated DNA sequencer asdescribed earlier (Heyrman and Swings 2001). The sequencing primers usedwere 5-AGHGTBTGHTCMTGNCTCAS-3 (Forward Primer) and5-TRCGGYTMCCTTGTWHCGACTH-3 (Reverse Primer). The amplified DNA fragmentof approximately 1.5 kb separated on a 1% agarose gel and purified byusing Qiagen spin columns. The purified fragment was used directly forDNA sequencing. This sequence was used in a BLAST search(http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Growth Conditions of Bacillus coagulans MTCC 25235

The optimization of growth conditions for Bacillus coagulans MTCC 25235was analyzed for different temperature and pH. MRSB media was preparedand adjusted pH to 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 with 2 NHCl and 2 N NaOH. Overnight grown culture (1%, v/v) inoculated to pHadjusted media and incubated for 24h in a shaking incubator at 120 rpm.Growth was monitored at every six hour interval of time by measuringabsorbance at 600 nm using spectrophotometer (Shimadzu Corporation,Kyoto, Japan). MRSB media was prepared adjusted pH to 6.5 and inoculatedwith overnight grown culture (1%, v/v). Further, flasks were incubatedfor 24h in shaking incubator at 120 rpm at different temperatures (20,30, 37, 40, 50 and 60° C.). Growth was monitored at every six hourinterval of time by measuring absorbance at 600 nm usingspectrophotometer (Shimadzu Corporation, Kyoto, Japan).

Results Identification of Fructophilic Bacteria

The spores of Bacillus coagulans MTCC 25235 were ellipsoidal terminalspores (FIG. 1A) and vegetative cells were Gram positive rod shaped asindicated in Gram's stain (FIG. 1B). Colonies from B. coagulans MTCC25235 were grown on media, yielding uniform, 1-3 mm in diameter, whiteto cream, smooth colonies that contain vegetative rod shaped cells(FIGS. 1C and D). The growth of the isolated bacteria was better infructose rich media rather than dextrose rich media (FIG. 2), indicatingits fructophilic nature and fructose utilization potential when comparedto other Bacillus coagulans strains. The growth of the bacterial wasalso tested in fructose rich food stuff and showed a deduction in thefructose content (FIG. 3) implying that the bacterium utilizes fructoseas a carbon source for its growth and development. Thus, the bacteriumcan be used in managing and preventing disorders related to highfructose intake by utilizing the excess fructose in the food stuff forits metabolism. The bacteria can be administered as a stand alone or incombination with foods rich in fructose which include high fructose cornsyrup, honey, Agave, Maple syrup, Coconut sugar, Palm sugar, Molasses,Soda, Candies, sweetened yogurt, frozen foods, canned foods, cereals,fruit juices, coffee creamer, jams and jellies, energy drinks,condiments, ice cream (Braverman J, List of Foods high in fructose.https://www.livestrong.com/article/30454-list-foods-high-fructose/,accessed 3 May 2019)

Biochemical Characterization

Biochemical characterization of Bacillus coagulans MTCC 25235 and B.coagulans ATCC 31284 was performed by the method described (Majeed, M.,Nagabhushanam, K., Natarajan, S., Sivakumar, A., Eshuis-de Ruiter, T.,Booij-Veurink, J., Janine Booij-Veurink, Ynte P. de Vries, Ali, F.(2016); Evaluation of genetic and phenotypic consistency of Bacilluscoagulans MTCC 5856: A commercial probiotic strain. World Journal ofMicrobiology & Biotechnology, 32,60). The results were compared withcommercial strain B. coagulans ATTCC 3128 and tabulated in Table 2.

TABLE 2 Biochemical characterization of B. coagulans ATTCC 3128 andBacillus coagulans MTCC 25235 B. coagulans B. coagulans S. No. TestsATTCC 3128 MTCC 25235 1. Catalase Positive Positive 2. Oxidase PositivePositive 3. Indole Negative Negative 4. Methyl Red Positive Positive 5.Voges Proskauers Positive Positive 6. Citrate Negative Negative 7. BloodHemolysis Negative Negative 8. Gram staining Gram Positive Gram Positive9. Lactose Positive Positive 10. Xylose Positive Positive 11. MaltosePositive Positive 12. Fructose Positive Positive 13. Dextrose PositivePositive 14. Galactose Positive Positive 15. Raffinose Positive Positive16. Trehalose Positive Positive 17. Melobiose Positive Positive 18.Sucrose Positive Positive 19. L-Arabinose Positive Positive 20. MannosePositive Positive 21. Inulin Positive Positive 22. Sodium gluconatePositive Positive 23. Glycerol Negative Negative 24. Salicin PositivePositive 25. Dulcitol Negative Negative 26. Inositol Negative Negative27. Sorbitol Positive Positive 28. Mannitol Positive Positive 29.Adonitol Positive Negative 30. Arabitol Positive Positive 31. ErythritolNegative Negative 32. α-Methyl-D- Positive Positive glucoside 33.Rhamnose Positive Positive 34. Cellobiose Positive Positive 35.Melezitose Negative Negative 36. α-Methyl-D- Negative Negative mannoside37. Xylitol Negative Negative 38. ONPG Positive Positive 39. Esculinhydrolysis Positive Positive 40. D-Arabinose Positive Positive 41.Citrate utilization Negative Negative 42. Malonate utilization NegativeNegative 43. Sorbose Negative Negative

The results indicated that the isolated probiotic strain is positive forbiochemical tests catalase, oxidase, methyl red, voges proskauers,lactose, xylose, maltose, fructose, dextrose, galactose, raffinose,trehalose, melobiose, sucrose, melobiose, arabinose, mannose, inulin,sodium gluconate, salicin, sorbitol, mannitol, arabitol, methylglucoside, rhamnose, cellobiose, ONPG, esculin hydrolysis and negativefor biochemical tests sorbose malonate utilization, citrate utilization,xylitol, methyl mannoside, melezitose, erythritol, adonitol, inositol,dulcitol, glycerol, blood hemolysis, citrate and indole.

16S rDNA Sequencing

The bacterial 16S rDNA was sequenced and the sequence information (SEQID) was obtained as below:

LOCUS  seq_FF7                 1437 bp ORIGIN   1 ACTTGCAAGT CGTGCGGCCC TTTTTTAAAA GCTTGCTTTT TAAAAGGTTA GCGGCGGACG  61 GGTGAGTAAC ACGTGGGCAC CCTGCCTGTA AGATCGGGAT AACGCCGGGA AACCGGGGCT 121 AATACCGGAT AGTTTTTTCC TCCGCATGGA GGAAAAAGGA AAGACGGCTT CTGCTGTCAC 181 TTACAGATGG GCCCGCGGCG CATTAGCTAG TTGGTGGGGT AACGGCTCAC CAAGGCAACG 241 ATGCGTAGCC GACCTGAGAG GGTGATCGGC CACATTGGGA CTGAGACACG GCCCAAACTC 301 CTACGGGAGG CAGCAGTAGG GAATCTTCCG CAATGGACGA AAGTCTGACG GAGCAACGCC 361 GCGTGAGTGA AGAAGGCCTT CGGGTCGTAA AACTCTGTTG CCGGGGAAGA ACAAGTGCCG 421 TTCGAACAGG GCGGCGCCTT GACGGTACCC GGCCAGAAAG CCACGGCTAA CTACGTGCCA 481 GCAGCCGCGG TAATACGTAG GTGGCAAGCG TTGTCCGGAA TTATTGGGCG TAAAGCGCGC 541 GCAGGCGGCT TCTTAAGTCT GATGTGAAAT CTTTGCGGGC TCACCCGCAA GCGGTCATTG 601 GAAACTGGGA GGGCTTTGAG TGCAAGAAAG AGGAGAGTGG AATTTCCACG TGTAGCGGTG 661 AAATGCGTAA AGATGTGGAG GAACACCAGT GGCGAAGGCG GCTCTCTGGT CTGTAACTGA 721 CGCTGAGGCG CGAAAGCGTG GGGAGCAAAC AGGATTAGAT ACCCTGGTAG TCCACGCCGT 781 AAACGATGAG TGCTAAGTGT TAGAGGGTTT CCGCCCTTTA GTGCTGCAGC TAACGCATTA 841 AGCACTCCGC CTGGGGAGTA CGGCCGCAAG GCTGAAACTC AAAGGAATTG ACGGGGGCCC 901 GCACAAGCGG TGGAGCATGT GGTTTAATTC GAAGCAACGC GAAGAACCTT ACCAGGTCTT 961 GACATCCTCT GACCTCCCTG GAGACAGGGC CTTCCCCTTC GGGGGACAGA GTGACAGGTG1021 GTGCATGGTT GTCGTCAGCT CGTGTCGTGA GATGTTGGGT TAAGTCCCGC AACGAGCGCA1081 ACCCTTGACC TTAGTTGCCA GCATTCAGTT GGGCACTCTA AGGTGACTGC CGGTGACAAA1141 CCGGAGGAAG GTGGGGATGA CGTCAAATCA TCATGCCCCT TATGACCTGG GCTACACACG1201 TGCTACAATG GATGGTACAA AGGGCTGCGA GACCGCGAGG TTAAGCCAAT CCCAGAAAAC1261 CATTCCCAGT TCGGATTGCA GGCTGCAACC CGCCTGCATG AAGCCGGAAT CGCTAGTAAT1321 CGCGGATCAG CATGCCGCGG TGAATACGTT CCCGGGCCTT GTACACACCG CCCGTCACAC1381 CACGAGAGTT TGTAACACCC GAAGTCGGTG AGGTAACCTT ACGGAGCCAG CCGCCGA //

A BLAST (Basic local alignment search tool) search was performed withthe above sequence and results of the first 10 alignment sequences aretabulated in table 3.

TABLE 3 Alignment view using combination of NCBI GenBank - Distributionof 10 Blast Hits on the Query Sequence Max Total Query S. No.Description score score cover E value Ident Accession 1 Bacilluscoagulans 2562 2562 100% 0.0 98.96% MF077122.1 strain 55-LR4 16Sribosomal RNA gene, partial sequence 2 Bacillus coagulans 2556 2556 100%0.0 98.89% MF992239.1 strain KCCM203098 16S ribosomal RNA gene, partialsequence 3 Bacillus sp. MC-02 2556 2556  99% 0.0 98.95% AB849115.1 genefor 16S ribosomal RNA, partial sequence 4 Bacillus coagulans gene 25562556 100% 0.0 98.89% AB696800.1 for 16S ribosomal RNA, partial sequence,strain: NTUIOB YUN2 5 Bacillus coagulans gene 2556 2556 100% 0.0 98.89%AB240205.1 for 16S rRNA, strain: T5 6 Bacillus coagulans 2553 2553 100%0.0 98.82% MH392659.1 strain ICMP 22322 16S ribosomal RNA gene, partialsequence 7 Bacillus sp. IMM05 2553 2553 100% 0.0 98.82% FR727705.1partial 16S rRNA gene, strain IMM05 8 Uncultured Bacillus sp. 2551 2551100% 0.0 98.82% MG557779.1 clone Bco 16S ribosomal RNA gene, partialsequence 9 Bacillus coagulans 2529 2529 100% 0.0 98.54% CP009709.1 DSM 1= ATCC 7050, complete genome 10 Bacillus coagulans 2505 2505 100% 0.098.19% NR_115727.1 strain ATCC 7050 16S ribosomal RNA gene, partialsequence

The results indicated that the isolated organism is a new strain ofBacillus coagulans having 98.96% identity with Bacillus coagulans strain55-LR4. The results of the phylogenetic analysis also indicated that theorganism is a new strain of Bacillus coagulans (FIG. 4)

Optimum of the Growth Conditions for Fructophilic Bacteria

The optimum pH and temperature recorded for the growth of fructophilicbacteria was 7.5 and 40° C. respectively (FIG. 5 and FIG. 6)

Example 2

In vitro Probiotic Evaluation of Bacillus coagulans MTCC 25235

Resistance to Gastric Acid

The survival of Bacillus coagulans MTCC 25235 spores was studied byaddition of 1 ml of suspension into 100 ml of a sterile electrolytesolution (6.2 g/L NaCl, 2.2 g/L KCl, 0.22 g/L CaCl2, and 1.2 g/L NaHCO3)containing 0.01% lysozyme (Sigma-Aldrich) and 0.3% pepsin(Sigma-Aldrich) and incubated for 5 min. Further, pH was adjusted to1.5, 3, 4, 5, 6, 7 and 8 (adjusted using 1 N NaOH and 1 N HCl).Incubation temperature was monitored to 37° C. for 4 h. One ml of samplewas withdrawn at different time intervals at 0, 1.0, 2.0, 3.0 and 4.0 h.After incubation, serial dilution was done in sterile saline (0.89% w/v)and the viable count was enumerated by plating on glucose yeast extractagar (HiMedia). Experiments were performed in triplicate at twodifferent occasions.

Bile Salt Tolerance

Bile tolerance of Bacillus coagulans MTCC 25235 cells was determined bythe method described earlier (Gilliland et al. 1984; Hyronimus et al.2000). MRS broth (HiMedia) was inoculated with approximately 10⁶ cfumL⁻¹ of Bacillus coagulans MTCC 25235 overnight grown culture and thensupplemented with (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5and 2.0%, w/v) bile salt and without bile salt as control in theexperiment. Samples were incubated for 24 h at 37° C. with shaking at120 rpm. Growth in control (no bile) and test cultures at differentconcentrations of bile was monitored hourly by measuring absorbance at600 nm using spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

Production of Lactic Acid

Lactic acid production by Bacillus coagulans MTCC 25235 was estimated byusing a Megazyme kit. A loopful of an overnight grown culture ofBacillus coagulans MTCC 25235 was added to glucose yeast extract broth(HiMedia) and incubated at 37° C. for 18 h with 120 rpm. Afterincubation, the broth was filtered through 0.22 micron (Sartorius,India) and analyzed for lactic acid content by using Megazyme kit(K-DLATE 10/04) as per instructions (Megazyme International Ireland, IDABusiness Park, Wicklow, Ireland). GYE media was taken as blank in theassay.

Simulated Gastric Juice Tolerance

The survival of Bacillus coagulans MTCC 25235 spores was studied torepresent buccal digestion conditions. One mL of the suspension ofBacillus coagulans MTCC 25235 was subjected to stimulated salivary juicecontents consist of KCl (0.8946 g/l), CO (NH₂)₂ (0.1981 g/L), Na₂SO₄(0.5681 g/L), NaHCO₃ (1.680 g/L), NaH₂PO₄ (0.8878 g/L), pH adjusted to6.8±0.2 and incubated at 37° C. for 5 min. Further, simulated gastricjuice was prepared by adding 9 g/L of sodium chloride and 3 g/L ofpepsin (Sigma-Aldrich, St. Louis, Mo., USA) and then the pH was adjustedto aseptically 3.0±0.2 by using 2 N HCl. Samples were further incubatedat 37° C. for 3 h with low r.p.m. After 3 h of incubation, pH wasadjusted aseptically to 7.0 using 2 N NaOH. Ox bile (5 g/L) this wasfurther incubated at 37° C. for 24 h. After final step of the simulateddigestion process, samples were collected and evaluated for the sporesurvival of Bacillus coagulans MTCC 25235 was enumerated by serialdilution method using glucose yeast extract agar media (HiMedia) (FIG.7).

Antibiotic Resistance Pattern

MIC was determined as per the guidelines of Clinical and LaboratoryStandards Institute (CLSI 2012). Bacillus coagulans MTCC 25235suspensions were prepared by suspending 18 h grown bacterial culture insterile normal saline (0.89% NaCl wt/vol; Himedia, Mumbai India). Theturbidity of the bacterial suspension was adjusted to 0.5 McFarlandstandards (equivalent to 1.5×10⁸ colony forming units (CFU)/m1). Theantibiotics stock solutions were prepared as per CLSI guidelines and2-fold serial dilutions were prepared in broth medium (glucose yeastextract acetate broth [GYEA, HiMedia, Mumbai India for Bacilluscoagulans MTCC 25235 and Mueller Hinton Broth [MHB, Difco Laboratories,Detroit, Mich. USA] for S. aureus) in 100 μl volume in 96-well U bottommicrotiter plates (BD Labware, N.J. USA). The above-mentioned bacterialsuspension was further diluted in the MHB and 100 μl volume of thisdiluted inoculum was added to each well of the plate resulting in thefinal inoculum of 5×10⁵ CFU/ml in the well and the final concentrationof antibiotics ranged from 0.0078 to 4 μg/ml. S. aureus ATCC 6538 wasused as reference culture in this study. The plates were incubated at37° C. for 24 h and were visually read for the absence or presence ofturbidity. The minimum concentration of the compound concentrationshowing no turbidity was recorded as MIC.

AMES Test

The data of the experiment suggested that the B. coagulans MTCC 25235spores did not increase the number of revertants in five Salmonellastrains (TA98, TA100, TA102, TA1535 and TA1537), compared with theirnegative controls, either absence or presence of the S9 metabolicactivation system. Further, no dose-dependent mutagenic effects werecaused by the B. coagulans MTCC 25235 spores (up to 5000 μg/plate). B.coagulans MTC 25235 spores did not show any mutagenic activity under theexperimental conditions.

BSH Activity

Bacillus coagulans MTCC 25235 growth was observed on an agar platecontaining ox bile and calcium carbonate, which indicates its toleranceagainst ox bile and the presence of BSH activity. There was 19±2 mmclear zone in the soft agar plate as shown in (FIGS. 8A and 8B) whichindicated the presence of BSH activity.

Antimicrobial Activity Against Human Pathogens

The antimicrobial activity was performed by a well diffusion assay aspreviously described with minor modifications (Cintas L M, Rodriguez JM, Fernandez M F, Sletten K, Nes I F, Hernandez P E, Holo H (1995)Isolation and characterization of pediocin L50, a new bacteriocin fromPediococcus acidilactici with a broad inhibitory spectrum. Appl EnvironMicrobiol 61:2643-2648). Briefly, a 5 mL lawn of soft (0.7% agar)glucose yeast extract (HiMedia, India), containing 10⁶ cfu mL⁻¹) of theindicator strains (S. epidermidis ATCC 14990, S. mutans MTCC 1943, S.aureus ATCC 29213, B. cereus ATCC 14579, P. acnes ATCC 11827, E. coliATCC 25922, P. aeruginosa ATCC 9027, M. luteus NCIM 216 and S. abonyNCIM 2257) was poured on top of an enriched hard (1.5% agar) trypticsoya agar (HiMedia, India). A loopful of overnight grown culture B.coagulans MTCC 25325 was added to MRS media and incubated for 24 h at37° C. with 120 rpm. After 24 h, the culture was centrifuged (10,000×9g) to remove the cells and the supernatant was collected, concentratedtenfold by lyophilization and filter-sterilized through a 0.22 micronfilter (Sartorius, India). Concentrated supernatant (50 μL) was added to6-mm wells punched in the solidified hi-layer agar. Plates were kept inthe refrigerator (4±2° C.) for 5 h to allow the sample to diffuse intothe agar and subsequently incubated at 37° C. for 18-20 h. Afterincubation, the zone of inhibition was measured and recorded in mm.

Production of Short Chain Fatty Acids

The in vitro fermentation with the Bacillus coagulans MTCC 25235 wascarried out by following method described by McBurney and Thompson(1987) with some modifications. Briefly, 2.0 g of fructose, fenugreekseeds, cranberry seed fiber, FOS, were added to 100 ml of demineralisedwater. The pH was adjusted to 7.0±0.2 and autoclaved at 121° C. for 20min. After sterilization, oxygen reducing enzyme Oxyrase (Oxyrase® forBroth, Oxyrase, Inc, Ohio, USA) was added to each flask, to induceanaerobic conditions. Five percent of overnight grown Bacillus coagulansMTCC 25235 culture was inoculated to all the flasks and incubated at 37°C. with gentle shaken rpm for 24 h. The bottles were tightly closed andsealed with parafilm to maintain anaerobic conditions generated by theenzyme supplement. The pH values at 0 h of incubation and afterfermentation (24 h) were also recorded. One ml of copper sulphate (10g/L) was added to each sample to inhibit further microbial growth(Sigma-Aldrich, St. Louis, Mo., USA). Further, 5.0 ml of samples wereadded to 5 ml of distilled water and pH was adjusted to 1.5 using 3 MH₂SO_(4.) 10 ml of chilled diethyl ether (−20° C.) was added to samplesand then vortex for 1 minute. Sodium chloride was added and thencentrifuged at 3000×g for 10 minutes. After centrifugation, organiclayer was separated and transferred to the fresh vial. This was used toquantify SCFAs. The SCFA standards were purchased from Sigma-Aldrich(St. Louis, Mo., USA) and similarly processed. SCFA production (acetate,propionate and butyrate) was measured by gas chromatography (GC) withthe use of a Agilent technologies 6890N gas chromatograph (Stevens CreekBlvd Santa Clara, Calif., USA) containing a DB-FFAP (Terephthalic acidmodified poly ethylene Glycol) column. The column temperature was 200°C. The injector and detector port temperatures were 250° C. The carriergas was N₂ at a flow rate of 1.0 ml/min. SCFA standards were purchasedfrom Sigma-Aldrich (St. Louis, Mo., USA). SCFA (Acetate, propionate andbutyrate) concentrations were expressed in mg/gram of galactomannan fromfenugreek seeds.

Result Resistance to Gastric Acid

There was no significant difference (2-5%) in spore count at pH 3 to pH8.0 in comparison to the initial spore count up to 4 h of the study(FIG. 9). However, 0.44 and 2.036 log₁₀ reduction was observed at pH 1.5in 1 and 4 h respectively. Results of the study confirmed the stabilityof Bacillus coagulans MTCC 25235 spores in acidic as well as alkaline pHconditions.

Bile Tolerance Test

Bacillus coagulans MTCC 25235 growth was observed on the agar platecontaining bile salt (1% w/v) which indicated its tolerance against bilesalt. Further, bile tolerance assay was performed by supplementing(0.1-2.0%) ox bile to the MRS broth in different flask. Bacilluscoagulans MTCC 25235 growth observed in presence and absence of ox bileup to 2%, w/v. Whereas, growth of B. coagulans ATCC 31284 was found in0.8% w/v (FIG. 10). Similarly, there was no significant difference inthe viability of Bacillus coagulans MTCC 25235 and B. coagulans ATCC31284 in the presence and absence of bile salt.

Production of Lactic Acid

Lactic acid production by Bacillus coagulans MTCC 25235 was estimated byusing a Megazyme kit. The total lactic acid produced by Bacilluscoagulans MTCC 25235 was 4.487 g/L. L-form of lactic acid was 4.12 g/L.Whereas, D-form of lactic acid Bacillus coagulans MTCC 25235 is 0.367g/L. (FIG. 11)

Antibiotic Resistance

MIC results of clindamycin, kanamycin, ampicillin, streptomycin,vancomycin, erythromycin, gentamicin, tetracycline and chloramphenicolagainst the Bacillus coagulans MTCC 25235 and S. aureus ATCC 6538 isgiven in table 4. All the antibiotics tested showed a MIC range of0.0078 to 1.0 μg/ml against Bacillus coagulans MTCC 25235. All testedantibiotics exhibited a MIC range of 0.031 to 2 μg/ml against S. aureusATCC 6538.

TABLE 4 Minimum inhibitory concentrations of antibiotics againstBacillus coagulans MTCC 25235 cultures and S. aureus ATCC 6538 MIC(μg/ml)* Bacillus coagulans S. aureus S. No. Antibiotics MTCC 25235 ATCC6538 1 Clindamycin hydrochloride 0.25 0.062 2 Kanamycin sulphate 0.0622.0 3 Ampicillin sodium salt 0.125 0.031 4 Streptomycin sulphate 0.0624.0 5 Vancomycin hydrochloride 0.25 0.5 6 Erythromycin 0.125 0.125 7Gentamicin sulphate 0.06 0.25 8 Tetracycline hydrochloride 0.03 0.062 9Chloramphenicol 0.031 2.0

Antimicrobial Activity

The antimicrobial activity of B. coagulans MTCC 25235 was evaluated.Anti-microbial activity was determined by well diffusion assay asdescribed by Cintas et al. (1995). The results indicated that theprobiotic bacteria is an effective antimicrobial agent againstSalmonella abonv, Micrococcus luteus, Escherichia coli, Pseudomonasaeruginosa, Bacillus cereus, Propionibacterium acnes, Streptococcusmutans, Staphylococcus aureus, Staphylococcus epidermidis.

TABLE 4 Antimicrobial activity of B. coagulans MTCC 25235 against testedbacteria S. no. Tested organism Zone of inhibition 1 Salmonella abonyNCIM 2257 14.00 ± 1.0 2 Micrococcus luteus NCIM 2169 18.50 ± 1.6 3Escherichia coli ATCC 25922 19.00 ± 2.0 4 Pseudomonas aeruginosa ATCC9027 17.42 ± 1.8 5 Bacillus cereus ATCC 14579 17.00 ± 2.1 6Propionibacterium acnes ATCC 11827 16.50 ± 1.2 7 Streptococcus mutansMTCC 1943  17.50 ± 1.75 8 Staphylococcus aureus ATCC 29213  15.85 ± 1.259 Staphylococcus epidermidis ATCC 14990  14.85 ± 1.40 Data represent themean ± SD of three independent experiments performed in triplicate

Short Chain Fatty Acid Production

The results of the analysis are presented in FIGS. 12A, 12B and 12C. Theproduction of acetic acid was high for B. coagulans MTCC 25235 with FOS,followed by fenugreek seed fibers, cranberry fibers and fructose (FIG.12A). Similarly, the production of butyric acid was high for B.coagulans MTCC 25235 with fructose, followed by FOS, fenugreek seedfibers and cranberry fibers (FIG. 12B). The production of propionic acidwas similar for B. coagulans MTCC 25235 cultured with fructose, FOS andcranberry seed fibers and lower for fenugreek seed fibers (FIG. 12C).

Storage and Viability

Two standardized preparations equivalent to 15×10⁹ (preparation 1) and6×10⁹ cfu/g (preparation 2) were studied. B. coagulans MTCC 25235 showedenhanced viability during the storage at temperature 40±2° C. with RH60%±5% (FIG. 13)

Other modifications and variations to the invention will be apparent tothose skilled in the art from the foregoing disclosure and teachings.Thus, while only certain embodiments of the invention have beenspecifically described herein, it will be apparent that numerousmodifications may be made thereto without departing from the spirit andscope of the invention. The scope of the invention is to be interpretedonly in conjunction with the appended claims.

We claim:
 1. A method for isolating and identifying novel fructoseutilizing probiotic bacteria from honey, comprising step of: a) Mixinghoney with saline in the ratio of 1:10 w/v to obtain a suspension; b)Thoroughly mixing the suspension of step a) and providing heat shock at50-70° C. for 30 minutes for selective isolation of spores; c) Isolatingthe bacterial colonies by incubating 1-2 ml of the suspension from stepb) in suitable culture media containing fructose for 48 hrs at 35-37°C.; d) Purifying bacterial isolates by selecting and incubatingmorphologically distinct colonies in a suitable media containingfructose as carbon source; e) Identifying the bacterial strain bybiochemical analysis and 16S rRNA sequencing as Bacillus coagulansstrain FF7 bearing accession no. MTCC
 25235. 2. The method as in claim1, wherein the honey is selected from the group comprising, raw honey,filtered honey, acacia honey, alfalfa honey, aster honey, avocado honey,basswood honey, beechwood honey, blueberry honey, bluegum honey,buckwheat honey, clover honey, dandelion honey, eucalyptus honey,fireweed honey, heather honey, ironbark honey, jarrah honey, leatherwoodhoney, linden honey, macadamia honey, manuka honey, orangeblossom honey,pinetree honey, sourwood honey, sage honey, and tupelo honey.
 3. Themethod as in claim 1, wherein the culture media is selected from thegroup comprising MRS (De Man, Rogosa and Sharpe agar), GYA (GlucoseYeast Extract Agar), TSB (Tryptone Soya Broth), Sporulation media andMueller Hinton Agar.
 4. The method as in claim 1, wherein the isolatedprobiotic strain returns positive for biochemical tests catalase,oxidase, methyl red, voges proskauers, lactose, xylose, maltose,fructose, dextrose, galactose, raffinose, trehalose, melobiose, sucrose,arabinose, mannose, inulin, sodium gluconate, salicin, sorbitol,mannitol, arabitol, methyl glucoside, rhamnose, cellobiose, ONPG,esculin hydrolysis and negative for biochemical tests sorbose malonateutilization, citrate utilization, xylitol, methyl mannoside, melezitose,erythritol, adonitol, inositol, dulcitol, glycerol, blood hemolysis,citrate and indole.
 5. A fructophilic probiotic bacteria of the genusBacillus, isolated from honey for increased utilization of fructose fromfoods rich in fructose.
 6. The probiotic bacteria as claimed in claim 5,wherein the said fructophilic probiotic bacterium is Gram positive. 7.The probiotic bacteria as claimed in claim 5, wherein the optimum pH andtemperature for the growth of fructophilic bacteria is 7.5 and 40° C.respectively.
 8. The probiotic bacteria as claimed in claim 5, whereinthe said fructophilic probiotic bacteria is bile tolerant, gastric acidresistance and produces lactic acid.
 9. The probiotic bacteria asclaimed in claim 5, wherein the said fructophilic probiotic bacterium isBacillus coagulans.
 10. The probiotic bacteria as claimed in claim 5,wherein the Bacillus coagulans strain is Bacillus coagulans MTCC 25235.11. The probiotic bacteria as claimed in claim 5, wherein the foods richin fructose are selected from the group comprising high fructose cornsyrup, honey, Agave, Maple syrup, Coconut sugar, Palm sugar, Molasses,Soda, Candies, sweetened yogurt, frozen foods, canned foods, cereals,fruit juices, coffee creamer, jams and jellies, energy drinks,condiments, ice cream.
 12. The probiotic bacteria as claimed in claim 5,wherein the said fructophilic probiotic bacteria is used for thetherapeutic management of disorders related to high fructose intake. 13.The disorders related to high fructose intake as in claim 12, areselected from the group comprising, obesity, non-alcoholicsteatohepatitis (NASH), insulin resistance, metabolic syndrome,cardio-vascular complications, diabetes, hyperlipidemia, hypertension,inflammation and hyperuricemia.
 14. The probiotic bacteria as claimed inclaim 5, wherein the said fructophilic probiotic bacteria is present inthe form of an inoculum, freeze-dried powder, fine powder, tablet,capsule, suspension, solution, emulsion, gummy, chewable or edible foodsand administered as a stand alone or in combination with foods rich infructose selected from the group comprising high fructose corn syrup,honey, Agave, Maple syrup, Coconut sugar, Palm sugar, Molasses, Soda,Candies, sweetened yogurt, frozen foods, canned foods, cereals, fruitjuices, coffee creamer, jams and jellies, energy drinks, condiments, icecream.
 15. A method of inhibiting pathogenic microbes said methodcomprising step of bringing to contact said microbes with thefructophilic probiotic bacteria Bacillus coagulans MTCC
 25235. 16. Themethod as in claim 15, wherein the pathogenic microbes are selected fromthe group comprising Salmonella abony, Micrococcus luteus, Escherichiacoli, Pseudomonas aeruginosa, Bacillus cereus, Propionibacterium acnes,Streptococcus nutans, Staphylococcus aureus, Staphylococcus epidermidis.17. A method of producing short chain fatty acids by culturing thefructophilic probiotic bacteria Bacillus coagulans MTCC 25235 along withplant fibers selected from the group consisting of fructose, fenugreekseed fibers, cranberry seed fibers, fructooligosaccharides (FOS). 18.The method as in claim 17, wherein the short chain fatty acids areselected from the group comprising acetic acid, butyric acid andpropionic acid.